Figure 5: Fat tissue filtrate stimulates the transformation of JB6 cells. A. JB6 cells were treated with 5% FBS or fat tissue filtrate (50 μg protein/mL) for 7 days, and then 104 cells were placed in soft agar for 14 days. The picture shows JB6 cells growing in soft agar that were cultured with fat tissue filtrate from mice fed either LFD or HFD (magnification = 400×). B. Cells were treated as described in (A). Fat tissue filtrate from mice fed a HFD stimulated colony formation in soft agar 160% more than fat pad filtrate from mice fed a LFD (*P<0.05). Data was obtained from 3 separate clones per treatment group. One dish = 20 cm2. All data are mean ± SEM, Student t test (unpaired, two-tailed). C. Mice were injected with 2x106 JB6 cells subcutaneously in the right and left flank of a SCID mouse. Mice were injected with cells treated with fat tissue filtrate as described in Figure 2. Data shows picture of tumor formation in SCID mice 10 days after injection. D. Immunohistochemistry of tumor in SCID mice injected with JB6 cells treated with HFD fat tissue filtrate demonstrates a fibrosarcoma at 40×and 200×magnification